Lung cancer is the leading cause of cancer related deaths in the United States accounting for approximately 160,000 deaths per year. Until recently, lung cancer has been classified and treated based on histological subtypes although currently more promising treatment strategies are being employed that involve assigning therapeutics based on alterations in the genome. The most successful molecular based therapeutics to date have been the tyrosine kinase inhibitors (TKIs) designed to target the epidermal growth factor receptor (EGFR). Challenges in targeting EGFR and other mutant proteins in lung cancer center largely around primary and acquired drug resistance although at the present time a complete understanding of mechanisms of resistance have not been fully elucidated. One common theory surrounding the maintenance of a TKI resistant state in lung cancer is the institution of compensatory kinases to maintain cell viability. One tyrosine kinase that has been shown to play a significant role in EGFR TKI resistance is EphA2. Preliminary data from our lab has shown that EphA2 is upregulated in many lung cancer cell lines and is further upregulated in EGFR TKI resistant cell lines. We have also shown that upon knockdown of EphA2, cell viability is inhibited in both lung cancer cell lines with innate resistance (i.e. K RAS mutations) and acquired resistance (i.e. EGFR T790M mutations) to EGFR TKIs. Because of significant evidence suggesting the utility of EphA2 as a novel target for inhibiting cell viabiity in cell populations refractory to EGFR TKI treatment, we propose the following aims to further dissect the role of EphA2 in TKI resistant lung cancer. In Aim 1, we will define the effects of targeting EphA2 in the context of a broad range of mutation signatures observed in lung cancer. In addition we will also test the benefits of combining EphA2 inhibition with additional kinase inhibitors for an enhanced loss of cell viability. In Aim 2, we will investigate the effects of targeting EphA2 in vivo by generating two mouse models to display EphA2 deficiency in the context of TKI resistance, Tet-O-EGFR(L858R+T790M)/EphA2 knockout and Tet-O-K-RAS(G12D)/EphA2 knockout mice. We will determine the effects of the loss of EphA2 on lung tumors in these mice by regular monitoring by MRI as well as immunohistochemical processing of lung tumor sections for proliferation and apoptosis. In Aim 3, we will examine the mechanism by which EphA2 controls cell viability in TKI resistant lung cancer cell populations. We will look for differences both in classical survival and proliferation signaling pathways as well as differences in metabolic activity between TKI resistant cell lines that are sensitive or resistant o EphA2 inhibition. Combined, this proposal will enable investigation into the role of EphA2 in innate or acquired resistance to EGFR TKIs and highlight the utility of using EphA2 as a novel target for lung cancers currently refractory to treatment.